Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.

Abstract

A long-term goal in the field of restrictionmodification enzymes has been to generate restriction endonucleases with novel sequence specificities by mutating or engineering existing enzymes. This will avoid the increasingly arduous task of extensive screening of bacteria and other microorganisms for new enzymes. Here, we report the deliberate creation of novel site-specific endonucleases by linking two different zinc finger proteins to the cleavage domain of Fok I endonuclease. Both fusion proteins are active and under optimal conditions cleave DNA in a sequence-specific manner. Thus, the modular structure of Fok I endonuclease and the zinc finger motifs makes it possible to create "artificial" nucleases that will cut DNA near a predetermined site. This opens the way to generate many new enzymes with tailor-made sequence specificities desirable for various applications.Since their discovery nearly 25 years ago (1), type II restriction enzymes have played a crucial role in the development of the recombinant DNA technology and the field of molecular biology. The type II restriction endonucleases and modification methylases are relatively simple bacterial enzymes that recognize specific sequences in duplex DNA. While the former cleave DNA, the latter methylate adenine or cytosine residues within the recognition site so as to protect the host genome against cleavage. So far, over 2500 restriction-modification (R-M) enzymes have been identified, and these are found in widely diverse organisms (2). These enzymes fall into numerous "isoschizomer" (identically cleaving) groups with about 200 sequence specificities. Discovery of new enzymes involves tedious and time-consuming effort that requires extensive screening of bacteria and other microorganisms (3). Even when one finds a new enzyme, more often than not, it falls into the already discovered isoschizomer groups. Furthermore, most naturally occurring restriction enzymes recognize sequences that are 4-6 bp long. Although these enzymes are very useful in manipulating recombinant DNA, they are not suitable for producing large DNA segments. For example, restriction enzymes that recognize DNA sequences 6 bp long result in cuts as often as every 4096 bases. In many instances, it is preferable to have fewer but longer DNA strands, especially during genome mapping. Rare cutters that recognize 8-bp-long sequences cut human DNA (which contains about 3 billion bp) every 65,536 bases on average. So far, only a few restriction endonucleases with recognition sequences longer than 6 bp (rare cutters) have been identified (New England Biolabs catalog). R-M systems appear to have a single biological function-namely, to protect cells from infection by foreign DNA that would otherwise destroy them. The phage genomes are usually small. It stands to reason, then, that bacteria select for R-M systems with small recognition sites (4-6 bp) because these sites occur more frequently in the phages. Therefore, aThe publication costs of this article were defrayed in part by page charge ...

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